All vertebrate kidney tissue is derived from the intermediate mesoderm (IM), a strip of tissue that lies between the somites and the lateral plate in the developing embryo. Three different types of kidney tissue are generated at particular levels along the anterior-posterior (A-P) embryonic axis--the pronephros, the mesonephros, and the metanephros. In addition, IM located anterior to the most anterior somites does not generate kidney tissue at all. The factors, which regulate IM differentiation along the A-P axis, are currently unknown. In order to gain insight into mechanisms of A-P patterning of the IM, the current proposal focuses on the problem of how the border at the 6th somite level between kidney-generating and non-kidney generating IM is formed. The avian embryo is used because it can be experimentally manipulated throughout the process of IM formation, allowing a broad range of in vivo and in vitro studies to be performed. The goal of the first aim is to determine when and where the anterior border of kidney gene expression is established. A series of in vivo transplant and in vitro explant studies will be performed, using quail and chick embryoninc tissues, and expression of the IM transcription factors lim-1, pax-2, and WT-1 will be examined using immunohistochemistry and in situ hybridization. The second aim examines the role of Hox genes in establishing the anterior border of kidney gene expression. Hox genes which are normally expressed only in the kidney-forming region of the IM will be expressed in more anterior regions using in vivo electroporation, and the effects on kidney gene expression will be examined. Expression of kidney markers will also be examined after lowering expression of these Hox genes through the use of antisense oligomers. The proposed investigations should advance our understanding of the molecular regulation of early kidney formation. This basic knowledge should aid efforts to generate kidney tissue in the laboratory for therapeutic purposes. The project will be a collaboration with the laboratory of Dr. Ram Reshef, Department of Biology, Technion-lsrael Instutute of Biology, Haifa, Israel, as an extension of NIH grant 5R01 DK59980-02.